Title

Author

Access Type

Open Access Dissertation

Date of Award

January 2017

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biomedical Engineering

First Advisor

Harini G. Sundararaghavan

Abstract

Peripheral nerve injuries affect millions of people each year around the world. Current treatments include an autograft, the gold standard, and commercially available nerve growth conduits (NGC). Autografts have several drawbacks including donor site morbidity and nerve size mismatch, which leads to incomplete recovery. Commercial NGCs can help with recovery but do not contain any specific cues to guide nerve regeneration. This thesis first evaluated mechanical, topographical and chemical cues that can be included in a NGC to promote and direct nerve regeneration. To incorporate all of the cues, a compliant substrate methacrylated hyaluronic acid (MeHA, mechanical cue) is electrospun into aligned fibers (topographical cue), with poly-lactic-co-glycolic acid (PLGA) microspheres to deliver growth factors (GF, chemical cue).

The properties of the scaffold were evaluated under physiological conditions using environmental scanning electron microscopy and mechanical testing in a physiological environment. The resulting scaffolds have hydrated porosities of 35-55% and young’s modulus from 0.43-2.86MPa. Nerve growth factor (NGF) was used as the GF for in vitro testing. The bioactivity of the encapsulated NGF was tested both during the short and long term. Results showed that NGF remains bioactive through the encapsulation and electrospinning process. ELISA showed that NGF is released from the microspheres for up to 4 weeks. Dorsal root ganglia (DRG) neurons were used to evaluate NGF bioactivity and testing showed that the released NGF was bioactive and could increase neurite outgrowth for up to 4 weeks. DRG testing on the scaffolds also showed that the combination of NGF released from the microspheres and the aligned nanofibers significantly directed and enhanced neurite outgrowth.

The study continued with in vivo testing by creating an NGC that included the scaffold as an inner support mechanism. An additional cue was then added in the form of treadmill running, to simulate physical therapy (PT). Circumferentially aligned nanofibers of Polycaprolactone (PCL) were layered with longitudinally aligned electrospun methacrylated Hyaluronic Acid (MeHA) fibers, with or without microspheres containing glial cell line-derived neurotrophic factor (GDNF), and rolled into conduits with an inner diameter of 1.25mm and length of 14mm. The conduits were implanted into an 8mm sciatic nerve gap in female Lewis rats. The animals were divided into five groups: fibers, fibers + PT, fibers + GF, fibers + GF + PT, and autograft control. All animals received the following behavior and functional testing prior to surgery and weekly post-surgery: Static Sciatic Index, Von Frey Filament Mechanical Sensory Test, and Ladder Walking Test. At the end of the study Compound Muscle Action Potentials (CMAP) and contractile force of the gastrocnemius muscle was measured. Sciatic nerves were harvested bilaterally for histological analysis. Weekly testing showed that fibers with GFs enhanced or sped functional recovery. Footfall measures the number of times an animal misses a rung when traversing a ladder, indicating gross muscle control. By 4 weeks both the GF groups were performing similarly to the autograft. Von Frey fibers were used to test the sensory perception. All groups showed hypersensitivity similar to the autograft by week 4, however the GF groups showed a response pattern similar to the autograft at all time points. Sciatic Index testing showed some improvement over the 8 weeks of testing. The groups receiving GDNF had greater improvement than those that did not. CMAP showed similar results.